Streptococcus pneumoniae is a Gram-positive bacteria responsible for considerable morbidity and mortality (particularly in the young and aged), causing invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonisation, such as acute Otitis media. The rate of pneumococcal pneumonia in the US for persons over 60 years of age is estimated to be 3 to 8 per 100,000. In 20% of cases this leads to bacteremia, and other manifestations such as meningitis, with a mortality rate close to 30% even with antibiotic treatment.
Pneumococcus is encapsulated with a chemically linked polysaccharide which confers serotype specificity. There are 90 known serotypes of pneumococci, and the capsule is the principle virulence determinant for pneumococci, as the capsule not only protects the inner surface of the bacteria from complement, but is itself poorly immunogenic. Polysaccharides are T-independent antigens, and can not be processed or presented on MHC molecules to interact with T-cells. They can however, stimulate the immune system through an alternate mechanism which involves cross-linking of surface receptors on B cells.
It was shown in several experiments that protection against invasive pneumococci disease is correlated most strongly with antibody specific for the capsule, and the protection is serotype specific.
Polysaccharide antigen based vaccines are well known in the art. Four that have been licensed for human use include the Vi polysaccharide of Salmonella typhi, the PRP polysaccharide from Haemophilus influenzae, the tetravalent meningococcal vaccine composed of serotypes A, C, W135 and Y, and the 23-Valent pneumococcal vaccine composed of the polysaccharides corresponding to serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33 (accounting for at least 90% of pneumococcal blood isolates).
The latter three vaccines confer protection against bacteria causing respiratory infections resulting in severe morbidity and mortality in infants, yet these vaccines have not been licensed for use in children less than two years of age because they are inadequately immunogenic in this age group [Peltola et al. (1984), N. Engl. J. Med. 310:1561-1566]. Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children. Likewise, the elderly mount poor responses to pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol. 42:265-270], hence the increased incidence of bacterial pneumonia in this population [Verghese and Berk, (1983) Medicine (Baltimore) 62:271-285].
Strategies, which have been designed to overcome this lack of immunogenicity in infants, include the linking of the polysaccharide to large immunogenic proteins, which provide bystander T-cell help and which induce immunological memory against the polysaccharide antigen to which it is conjugated. Pneumococcal glycoprotein conjugate vaccines are currently being evaluated for safety, immunogenicity and efficacy in various age groups.
A) Pneumococcal Polysaccharide Vaccines
The 23-valent unconjugated pneumococcal vaccine has shown a wide variation in clinical efficacy, from 0% to 81% (Fedson et al. (1994) Arch Intern Med. 154: 2531-2535). The efficacy appears to be related to the risk group that is being immunised, such as the elderly, Hodgkin's disease, splenectomy, sickle cell disease and agammaglobulinemics (Fine et al. (1994) Arch Intern Med. 154:2666-2677), and also to the disease manifestation. The 23-valent vaccine does not demonstrate protection against pneumococcal pneumonia (in certain high risk groups such as the elderly) and otitis media diseases.
There is therefore a need for improved pneumococcal vaccine compositions, particularly ones which will be more effective in the prevention or amelioration of pneumococcal disease (particularly pneumonia) in the elderly and in young children.
The present invention provides such an improved vaccine.
B) Selected Pneumococcal Polysaccharide Conjugate+3D-MPL Compositions
It is generally accepted that the protective efficacy of the commercialised unconjugated pneumococcal vaccine is more or less related to the concentration of antibody induced upon vaccination; indeed, the 23 polysaccharides were accepted for licensure solely upon the immunogenicity of each component polysaccharide (Ed. Williams et al. New York Academy of Sciences 1995 pp. 241-249). Therefore further enhancement of antibody responses to the pneumococcal polysaccharides could increase the percentage of infants and elderly responding with protective levels of antibody to the first injection of polysaccharide or polysaccharide conjugate and could reduce the dosage and the number of injections required to induce protective immunity to infections caused by Streptococcus pneumoniae. 
Since the early 20th century, researchers have experimented with a huge number of compounds which can be added to antigens to improve their immunogenicity in vaccine compositions [reviewed in M. F. Powell & M. J. Newman, Plenum Press, NY, “Vaccine Design—the Subunit and Adjuvant Approach” (1995) Chapter 7 “A Compendium of Vaccine Adjuvants and Excipients”]. Many are very efficient, but cause significant local and systemic adverse reactions that preclude their use in human vaccine compositions. Aluminium-based adjuvants (such as alum, aluminium hydroxide or aluminium phosphate), first described in 1926, remain the only immunologic adjuvants used in human vaccines licensed in the United States.
Aluminium-based adjuvants are examples of the carrier class of adjuvant which works through the “depot effect” it induces. Antigen is adsorbed onto its surface and when the composition is injected the adjuvant and antigen do not immediately dissipate in the blood stream—instead the composition persists in the local environment of the injection and a more pronounced immune response results. Such carrier adjuvants have the additional known advantage of being suitable for stabilising antigens that are prone to breakdown, for instance some polysaccharide antigens.
3D-MPL is an example of a non-carrier adjuvant. Its full name is 3-O-deacylated monophosphoryl lipid A (or 3 De-O-acylated monophosphoryl lipid A or 3-O-desacyl-4′ monophosphoryl lipid A) and is referred to as 3D-MPL to indicate that position 3 of the reducing end glucosamine is de-O-acylated. For its preparation, see GB 2220211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. It was originally made in the early 1990's when the method to 3-O-deacylate the 4′-monophosphoryl derivative of lipid A (MPL) led to a molecule with further attenuated toxicity with no change in the immunostimulating activity.
3D-MPL has been used as an adjuvant either on its own or, preferentially, combined with a depot-type carrier adjuvant such as aluminium hydroxide, aluminium phosphate or oil-in-water emulsions. In such compositions antigen and 3D-MPL are contained in the same particulate structures, allowing for more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further enhance the immunogenicity of an alum-adsorbed antigen [Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1]. Such combinations are also preferred in the art for antigens that are prone to adsorption (for instance, bacterial polysaccharide conjugates), where adsorption onto alum tends to stabilise the antigen. Precipitated aluminium-based adjuvants are mostly used as they are the only adjuvants that are currently used in licensed human vaccines. Accordingly, vaccines containing 3D-MPL in combination with aluminium-based adjuvants are favoured in the art due to their ease of development and speed of introduction onto the market.
MPL (non 3-deacylated) has been evaluated as an adjuvant with several monovalent polysaccharide-conjugate vaccine antigens. Coinjection of MPL in saline enhanced the serum antibody response for four monovalent polysaccharide conjugates: pneumococcal PS 6B-tetanus toxoid, pneumococcal PS 12-diphtheria toxoid, and S. aureus type 5 and S. aureus type 8 conjugated to Pseudomonas aeruginosa exotoxin A [Schneerson et al. J. Immunology (1991) 147:2136-2140]. The enhanced responses were taught as being antigen-specific. MPL in an oil-in-water emulsion (a carrier type adjuvant) consistently enhanced the effect of MPL in saline due to the presence of MPL and antigen in the same particulate structure, and was considered to be the adjuvant system of choice for optimal delivery of other polysaccharide conjugate vaccines.
Devi et al. [Infect. Immun. (1991) 59:3700-7] evaluated the adjuvant effect of MPL (non 3-deacylated) in saline on the murine antibody response to a TT conjugate of Cryptococcus neoformans capsular polysaccharide. When MPL was used concurrently with the conjugate there was only a marginal increase in both the IgM- and IgG-specific response to the PS; however MPL had a much larger effect when administered 2 days after the conjugate. The practicality of using an immunization scheme that requires a delay in the administration of MPL relative to antigen, especially in infants, is questionable. The adjuvant effect of MPL with polysaccharides and polysaccharide-protein conjugates appears to be composition-dependent. Again, the incorporation of MPL in a suitable slow-release delivery systems (for instance using a carrier adjuvant) provides a more durable adjuvant effect and circumvents the problem of timing and delayed administration.
In summary, the state of the art has taught that, for particular polysaccharide or polysaccharide-conjugate antigens, where MPL or 3D-MPL is used as an adjuvant, it is advantageously used in conjuction with a carrier adjuvant (for instance the aluminium-based adjuvants) in order to maximise its immunostimulatory effect.
Surprisingly, the present inventors have found that for certain pneumococcal polysaccharide conjugates, the immunogenicity of the vaccine composition is significantly greater when the antigen is formulated with 3D-MPL alone rather than with 3D-MPL in conjunction with a carrier adjuvant (such as an aluminium-based adjuvant). Furthermore the observed improvement is independent of the concentration of 3D-MPL used, and whether the particular conjugates are in a monovalent composition or whether they are combined to form a polyvalent composition.
C) Bacterial Polysaccharide—Protein D Conjugates
As mentioned above, polysaccharide antigen based vaccines are well known in the art. The licensed polysaccharide vaccines mentioned above have different demonstrated clinical efficacy. The Vi polysaccharide vaccine has been estimated to have an efficacy between 55% and 77% in preventing culture confirmed typhoid fever (Plotkin and Cam, (1995) Arch Intern Med 155: 2293-99). The meningococcal C polysaccharide vaccine was shown to have an efficacy of 79% under epidemic conditions (De Wals P, et al. (1996) Bull World Health Organ. 74: 407-411). The 23-valent pneumococcal vaccine has shown a wide variation in clinical efficacy, from 0% to 81% (Fedson et al. (1994) Arch Intern Med. 154: 2531-2535). As mentioned above, it is accepted that the protective efficacy of the pneumococcal vaccine is more or less related to the concentration of antibody induced upon vaccination.
Amongst the problems associated with the polysaccharide approach to vaccination, is the fact that polysaccharides per se are poor immunogens. Strategies which have been designed to overcome this lack of immunogenicity include the linking of the polysaccharide to large highly immunogenic protein carriers, which provide bystander T-cell help.
Examples of these highly immunogenic carriers which are currently commonly used for the production of polysaccharide immunogens include the Diphtheria toxoid (DT or the CRM197 mutant), Tetanus toxoid (TT), Keyhole Limpet Haemocyanin (KLH), and the purified protein derivative of Tuberculin (PPD).
Problems Associated with Commonly-Used Carriers
A number of problems are associated with each of these commonly used carriers, including in production of GMP conjugates and also in immunological characteristics of the conjugates.
Despite the common use of these carriers and their success in the induction of anti polysaccharide antibody responses they are associated with several drawbacks. For example, it is known that antigen specific immune responses may be suppressed (epitope suppression) by the presence of preexisting antibodies directed against the carrier, in this case Tetanus toxin (Di John et al; (1989) Lancet, 2:1415-8). In the population at large, a very high percentage of people will have pre-existing immunity to both DT and TT as people are routinely vaccinated with these antigens. In the UK for example 95% of children receive the DTP vaccine comprising both DT and TT. Other authors have described the problem of epitope suppression to peptide vaccines in animal models (Sad et al, Immunology, 1991; 74:223-227; Schutze et al, J. Immunol. 135: 4, 1985; 2319-2322).
In addition, for vaccines which require regular boosting, the use of highly immunogenic carriers such as TT and DT are likely to suppress the polysaccharide antibody response after several injections. These multiple vaccinations may also be accompanied by undesirable reactions such as delayed type hyperresponsiveness (DTH).
KLH is known as potent immunogen and has already been used as a carrier for IgE peptides in human clinical trials. However, some adverse reactions (DTH-like reactions or IgE sensitisation) as well as antibody responses against antibody have been observed.
The selection of a carrier protein, therefore, for a polysaccharide based vaccine will require a balance between the necessity to use a carrier working in all patients (broad MHC recognition), the induction of high levels of anti-polysaccharide antibody responses and low antibody response against the carrier.
The carriers used previously for polysaccharide based vaccines, therefore have many disadvantages. This is particularly so in combination vaccines, where epitope suppression is especially problematic if the same carrier is used for various polysaccharide antigens. In WO 98/51339, multiple carriers in combination vaccines were used in order to try to get over this effect.
The present invention provides a new carrier for use in the preparation of polysaccharide/polypeptide-based immunogenic conjugates, that does not suffer from the aforementioned disadvantages.
The present invention provides a protein D (EP 0 594 610 B1) from Haemophilus influenzae, or fragments thereof, as a carrier for polysaccharide based immunogenic compositions, including vaccines. The use of this carrier is particularly advantageous in combination vaccines.